7K34

Crystal structure of L-threonine transaldolase from Pseudomonas fluorescens in internal aldimine state

Summary for 7K34

• Entry DOI: 10.2210/pdb7k34/pdb
• Descriptor: Threonine aldolase, SULFATE ION (3 entities in total)
• Functional Keywords: threonine transaldolase, pyridoxal phosphate, plp, biocatalysis, biosynthetic protein, synthase
• Biological source: Pseudomonas fluorescens
• Total number of polymer chains: 4
• Total formula weight: 201843.91

Authors

Kumar, P.,Bingman, C.A.,Buller, A.R. (deposition date: 2020-09-10, release date: 2020-12-30, Last modification date: 2023-11-15)

Primary citation

Kumar, P.,Meza, A.,Ellis, J.M.,Carlson, G.A.,Bingman, C.A.,Buller, A.R.
l-Threonine Transaldolase Activity Is Enabled by a Persistent Catalytic Intermediate.
Acs Chem.Biol., 16:86-95, 2021

PubMed Abstract: l-Threonine transaldolases (lTTAs) are a poorly characterized class of pyridoxal-5'-phosphate (PLP) dependent enzymes responsible for the biosynthesis of diverse β-hydroxy amino acids. Here, we study the catalytic mechanism of ObiH, an lTTA essential for biosynthesis of the β-lactone natural product obafluorin. Heterologously expressed ObiH purifies as a mixture of chemical states including a catalytically inactive form of the PLP cofactor. Photoexcitation of ObiH promotes the conversion of the inactive state of the enzyme to the active form. UV-vis spectroscopic analysis reveals that ObiH catalyzes the retro-aldol cleavage of l-threonine to form a remarkably persistent glycyl quinonoid intermediate, with a half-life of ∼3 h. Protonation of this intermediate is kinetically disfavored, enabling on-cycle reactivity with aldehydes to form β-hydroxy amino acids. We demonstrate the synthetic potential of ObiH via the single step synthesis of (2,3)-β-hydroxyleucine. To further understand the structural features underpinning this desirable reactivity, we determined the crystal structure of ObiH bound to PLP as the Schiff's base at 1.66 Å resolution. This high-resolution model revealed a unique active site configuration wherein the evolutionarily conserved Asp that traditionally H-bonds to the cofactor is swapped for a neighboring Glu. Molecular dynamics simulations combined with mutagenesis studies indicate that a structural rearrangement is associated with l-threonine entry into the catalytic cycle. Together, these data explain the basis for the unique reactivity of lTTA enzymes and provide a foundation for future engineering and mechanistic analysis.

PubMed: 33337128
DOI: 10.1021/acschembio.0c00753

Experimental method

X-RAY DIFFRACTION (1.66 Å)